Immortalized avian cell lines

ABSTRACT

The invention features non-transformant immortal avian cells, in particular derived from avian tissues, i.e., other than blood or hematopoietic cells, particularly fibroblasts and epithelial cells, for instance of embryos. The avian cells are immortalized by the SV40 T+t gene in the dependence of the MTI promoter. In particular they integrate the pDAMT vector.

This is a continuation of U.S. patent application Ser. No. 09/194,020filed on Jun. 23, 1999, now issued as U.S. Pat. No. 6,280,970 which is a371 of PCT/FR97/99898 filed May 22, 1997.

The present invention relates to an avian cell line and its derivatives.

It is not possible to establish cell lines spontaneously from organstaken from avian species, as can be done in the case of some organsderived from mammalian species.

The only available cell lines to date were obtained using thetransforming properties of certain avian viruses which possess oncogenicproperties, such as the retroviruses of the avian leukosis group orMarek's disease virus, or certain chemical molecules such asmethylcholanthrene and diethylnitrosamine.

For the most part, these cell lines are considerably transformed, whichrenders them unsuitable for multiplying vaccine viruses.

Authors are adopting a novel approach which consists in introducing intocells a vector which does not exhibit any oncogenic character but whichis able to integrate, into these cells, a gene which is selected for itscapacity to induce immortalization.

The first tests were carried out using vectors which integrate avianretrovirus genes such as erbA and erbB.

French Patent Application FR-A-2 596 770 proposes an immortalizationmethod in which a culture of avian or mammalian cells is infected with avector or a system which, while not being oncogenic for the said cells,is able to integrate a gene selected from v-myb, v-ets and v-erbA intothese cells. The AMV, E26 and XJ12 viruses, with this latter being avirus derivative of the AEV virus in which the oncogenic v-erB gene hasbeen deleted, can be appropriate vectors.

In practice, while these tests made it possible to obtain establishedcell lines from cells of the haematopoietic cell line, they did not givethe expected results in the case of chick embryo cells in adherentculture, such as fibroblasts or epithelial cells.

It was possible to obtain untransformed avian cell lines of themyeloblastoid type (blood cells) using the oncogene myb (InternationalPatent Application WO91/18971).

In parallel, authors have proposed using the early t and T genes of thesimian virus SV40 for immortalizing cells derived from differentmammalian tissues (D. S. Neufeld et al., Molecular and Cellular Biology,August 1987, 2794-2802, O. Kellermann and F. Kelly, Differentiation1986, 32:74-81 and French Patent Application FR-A-2 649 721).

For its part, French Patent Application FR-A-2 649 721 proposes a methodof conditional immortalization which, it is claimed, can be used for anycell type and in any species, with the aim in this case being that ofremedying the drawback of the high degree of specificity of theconventional approaches (limitation to particular species and/or toparticular cell types): transformation of cells with a transformingvirus (adenovirus, Epstein-Barr virus, certain papovaviruses such as theSV40 virus or polyoma virus; for example, the SV40 virus is indicated asonly transforming rodent cells and human cells); transfection withconstructs which contain a transforming gene which is linked to a viralpromoter; transfection with a transforming gene which is linked to acellular promoter. The choice of this patent application falls on aconstruct which combines a DNA fragment from the regulatory sequence ofvimentin and a DNA fragment which encodes an immortalizing gene, whichconstruct can be the T antigen of the SV40 virus under the control ofthe inducible promoter of vimentin. This document never mentions theavian species.

The actual use of such viral oncogenes has never been described in theavian species, apart from the use of the 12S form of the E1A protein ofhuman adenovirus 5, which made it possible to immortalize quailepithelial cells (Guilhot et al. (1993), Oncogene 8:619-624).

Contrary to all expectation, the inventors succeeded in producing animmortal, untransformed avian cell line.

More generally, the inventors have found that it was possible to prepareimmortal, untransformed avian cell lines even from cells of aviantissues, that is to say from cells other than circulating blood cells orhaematopoietic cells.

The present invention therefore relates to the immortal, untransformedavian cells which derive, in particular, from avian tissues, that is tosay from cells other than blood cells or haematopoietic cells, inparticular fibroblasts and epithelial cells, for example from embryos.

The present invention relates, in particular, to immortal, untransformedavian cells which contain, integrated into their genome, the SV40 T+tgene under the control of the MTI (murine metallothionein I) promoter.

Preferably, the cells also integrate the SV40 promoter, which isfunctionally linked to the gene for resistance to neomycin.

Preferably, the cells also integrate at least one LTR sequence. The LTRsequence can be deleted as described in the examples.

The cells preferably integrate the vector pDAMT which is depicted inFIG. 1.

While the cells are of avian origin, they may in particular be derivedfrom Muscovy duck.

The invention relates, more especially, to the immortal, untransformedavian cell line TDF-2A, which is deposited in the CNCM (CollectionNationale de Cultures de Microorganismes de l'Institut Pasteur (PasteurInstitute National Collection of Microorganism Cultures)) underreference number I-1712.

The invention naturally covers the cells which are derived from thesecell lines. By this, it is to be understood that it is not only thecells as deposited in the CNCM under the indicated references which arecovered but also the cells which constitute the progeny of thesedeposited cells, i.e., on the one hand, those which are obtained bysimple multiplication and which may undergo mutations during thesemultiplications and, on the other hand, those which are obtained afterdeliberate modification, which are then termed derived cells, and, ofcourse, also those which have undergone the two types of modification.

The invention therefore also covers the derived cells which are obtainedby modifications of the above cells. These modifications may consist in:

-   -   Inserting one or more expression cassettes, each of which        comprises one or more nucleotide sequences encoding a molecule        of industrial relevance, with these expression cassettes being        able to produce this molecule following insertion into the cells        of the invention. The skilled person is fully conversant with        the technique. Molecules of industrial relevance which may be        mentioned, in particular, are viral subunits of the peptide,        protein or glycoprotein type, in particular for use in a vaccine        or a diagnostic reagent, protein molecules such as hormones,        etc.    -   Chronically infecting with a virus which is able to multiply in        the cells, for virus or vaccine production purposes, with or        without prior modification of the sensitivity towards this        virus. The infection may also not be chronic but carried out on        a batch of cells which is selected for the viral multiplication.        (The modifications described below are to be understood as        preferably and advantageously being combined with the preceding        two types of modification).    -   Introducing survival or anti-apoptotic genes other than bcl-2,        such as the genes which encode the human adenovirus p19E1B (Rao        et al. (1992), Proc. Natl. Acad. Sci. USA 89:7742-7746), the        Epstein Barr virus LMP-1 (Gregory et al. (1991), Nature        349:612-614) and BHRF1 (Pearson et al. (1987), Virology        160:151-161), the herpes simplex virus ICP34.5 (Chou and Roizman        (1992), Proc. Natl. Acad. Sci. USA 89:3266-3270) and the        baculovirus p35 (Clem et al. (1991), Science 254:1388-1390)        proteins in order to render these cell lines more resistant to        the culture conditions, in particular for maintaining        confluence.    -   Overexpressing genes which are involved in controlling the cell        cycle using vectors which are suitable for increasing the rate        of proliferation. Thus, it has been demonstrated that, in        certain cases, overexpressing cyclin-encoding genes leads to the        cell cycle being shortened and therefore to the rate of        proliferation being increased (Rosenberg et al. (1995), Oncogene        10:1501-1509; Quelle et al. (1993), Genes and Dev. 7:1559-1571).    -   Modifying the viral sensitivity spectrum of the cell lines by        integrating genes which encode receptors for the viruses of        interest, with a view to multiplying these viruses.        Reference may be made to the mammalian species, where expression        of the receptor for the measles virus (CD46) by murine cells,        which are normally non-permissive for the virus, results in        these cells becoming sensitive to this virus and being able to        replicate it (Naniche et al. (1993), J. Virol. 67:6025-6032).        The interest is, in particular, in rendering cells sensitive to        a virus in order to produce the virus on these cells.    -   Integrating oncogenes which are able to accelerate cell growth.

It is self-evident that the derived cells according to the invention maycomprise one or more of the above-described modifications.

The invention also relates to a method for producing molecules ofindustrial relevance or viruses, which method comprises culturing theabove-described cells.

The present invention is directed, in particular, towards producingmolecules or viruses for creating diagnostic reagents or vaccines, orelse towards producing molecules of therapeutic relevance.

BRIEF DESCRIPTION OF THE DRAWING

The invention will now be described in more detail with the aid of anembodiment which is taken by way of non-limiting example and withreference to FIG. 1, which shows the structure of the vector pDAMT,which

-   is used to prepare the cell line TDF-2A, and in which:-   LTR:direct repeat sequence (long terminal repeat)-   δLTR:deleted LTR-   MTI:murine metallothionein I promoter-   SV40 T+t:SV40 early region-   SV40:SV40 promoter

EXAMPLE 1=PRODUCTION OF THE TDF-2A CELL LINE I. Description of itsOrigin and its Characteristics

1.1 Description of the Vector Employed:Vector pDAMT

It comprises the SV40 virus early region (encodes the T and t antigens)(HindIII/BamHI fragment) (Fiers et al. (1978), Nature 273:113-120) underthe control of the mouse metallothionein I promoter (EcoRI/BglIIfragment with the BglII site being transformed into a HindIII site)(Durnam et al. (1980), Proc. Natl. Acad. Sci. USA 77:6511-6515; Brinsteret al. (1982), Nature 296:39-42).

The EcoRI/EcoRI fragment containing this transcription unit, derivedfrom the vector pMTSVneo (Peden et al. (1989), Exp. Cell. Res.185:60-72), was inserted into the XbaI site of the vector pDA1 (Aubertet al. (1991), J. Cell. Biol. 113:497-506). This latter vector isessentially derived from the genome of the Rous sarcoma-associated virus2 (RAV-2) following modification of the 3′ LTR. Thus, the U3 region ofthe RAV-2 3′ LTR was deleted and linked to the R and U5 regions isolatedfrom the Rous sarcoma-associated virus 1 (RAV-1) LTR. The vector alsocarries a transcription unit which contains the gene for resistance toneomycin under the control of the SV40 promoter derived from the vectorpSV2neo (Southern and Berg (1982), J. Mol. Appl. Genet. 1:327-341). SeeFIG. 1.

1.2. Establishment of the Cell Line and Demonstration that it isImmortalized

Cells derived from 14-day Muscovy duck embryos were transfected withvector pDAMT using the dimethyl sulphoxide (DMSO) method described byKawai and Nishizawa (1984), Mol. Cell. Biol. 4:1172-1174. Thetransfected cells are then selected by applying geneticin G418 (150μg/ml) for 15 days. The resistant clones are then subcultured regularlyat the rate of from 1 to 2 passages per week. After this 3-month periodof active proliferation, the cells entered into a crisis period duringwhich most of the cells died. After this period, which lastedapproximately 2 months, several clones resumed active proliferation,suggesting that they had been immortalized.

The TDF-2A cell line is thus derived from 2 cultures.

It was studied in more depth.

The TDF-2A cells achieved 200 passages, that is approximately 460generations, and were thus maintained continuously in culture for morethan 600 days. By comparison, control cells, which are not expressingthe SV40 virus early region, cannot be maintained in culture for morethan 20 passages.

1.3. Proliferation Characteristics

The immortalized cells are cultured at 38° C., in a roller bottle, in amedium containing 6% 10× HAM F-10, 4% 10×199 HANKS, from 2.95% to 4%tryptose broth phosphate, from 5.6% to 2.5% sodium bicarbonate, 0.1%100×vitamin BME, 3% foetal calf serum, from 5% to 1% kanamycin and from0.5% to 1% vancomycin.

Under these conditions, their rate of doubling is once every 24 hours.

1.4. Expression of the T Antigen

It was verified, by means of indirect immunofluorescence or indirectimmunophosphatase using an antibody which is specific for the T antigen(Pab 101:Santa Cruz Biotechnology ref. sc147), that all the cellsexpress the T antigen in their nucleus, indicating that they have allintegrated the vector.

This integration was additionally demonstrated by means of Southernblotting. The genomic DNA of the immortalized fibroblasts was digestedwith the restriction enzymes XbaI and BstXI. Hybridization with a probewhich was specific for the T antigen (1018 bp NdeI/NdeI fragment)verified that the transcription unit, which expressed the immortalizinggene and which was inserted into the TDF-2A cells, had not undergone anymajor rearrangements. This was indicated by the fact that the sizes ofthe hybridization fragments obtained were in accordance with theexpected sizes.

1.5. Absence of Tumorigenic Capacity

The immortalized cells do not exhibit any tumorigenic capacity. They areincapable of forming colonies in semi-solid medium or of forming tumourson hen or duck egg chorioallantoic membrane. They are also incapable offorming tumours on nude mice, and one-day old SPF (pathogen-free)ducklings and chicks.

1.6. Karyotype

The karyotype of the TDF-2A cells was studied at the 114th and 135thpassages. This verified that the cells were indeed of avian origin, withthe microchromosomes characteristic of this species being present.Furthermore, the chromosomes which were observed are representative ofthe chromosomes which are encountered in primary duck embryo cells,thereby confirming the origin of the cell line.

II. Properties

The TDF-2A cells exhibit, in particular, a sensitivity to theduck-specific viruses, such as adenovirus, parvovirus and retrovirus,which are customarily replicated on primary duck embryo cells. Theseviruses can therefore be produced on this cell line.

EXAMPLE 2 Characterization of the TDF-2A Cell Line by Identifying theIntegration Sites

The genomic DNA of the TDF-2A cells, which was prepared from cellsderived from the 114th and 135th passages, was digested with therestriction enzymes BglII and KpnI. The DNA, which had been treated inthis way, was then subjected to gel electrophoresis, followed bytransfer to a nylon membrane; it was then hybridized with a probe whichwas specific for the T antigen (1018 bp NdeI/NdeI fragment). Forexample, digestion with BglII gives rise to two hybridization bands oflarge size (approximately 15 and 23 kb), suggesting the existence of twointegration sites. Digestion with KpnI gives rise to one major band-oflarge size (approximately 20 kb) and to at least one minor band, therebyconfirming the existence of at least two integration sites.

EXAMPLE 3 Multiplication of Adenovirus V127 on TDF-2A Cells

The TDF-2A cells are seeded in a roller bottle. Soft-shelled egg diseaseadenovirus strain V127 is inoculated into the cell culture. After 6days, harvesting is carried out by shaking in order to detach the celllawn. The harvested mixture therefore consists of the cell lawn and theculture supernatant. The whole is homogenized by treating with a cellgrinder or homogenizer such as Ultraturrax for 1 min at 13,500 rpm(T25-type IKA appliance).

The infectious viral titre is determined by means of a micromethodcarried out on 96-well plates. The virus dilutions are inoculated onto alawn prepared from secondary SPF Muscovy duck embryo cells. Each viraldilution is inoculated into 6 wells. The plates are incubated in a CO₂incubator for 8 days. The presence of the virus in the wells is checkedby observing the characteristic cytopathic effect (CPE) under themicroscope. The infectious titre is calculated by the KARBER method andis expressed by the logarithm of the inverse of the viral dilution whichgives 50% CPE [titre=d+r/Nx(n+N/2)], where d is the dilution expressedin logs when all the wells are positive, r is the dilution ratio, N isthe number of wells per dilution and n is the number of positive wellsbetween 0 and 100%.

The presence of the virus is also confirmed by investigating thehaemagglutinating activity of the viral supernatant using a suspensionof chick red blood corpuscles. In this case, 50 μl of supernatant fromthe abovementioned wells are deposited on a Dynatech microtitrationplate and 25 μl of a suspension of chick erythrocytes containing 15.10⁶cells/ml are added per well. After the microtitration plate has beenshaken and incubated at ambient temperature for 45 min, all the wellswhich exhibit clearly visible haemagglutination are regarded as beingpositive. The titre is likewise calculated by the KARBER method.

Results: The viral titres which are obtained are equivalent to thoseobtained on primary duck embryo cells.

1. A method for producing viruses, said method comprising: infecting anavian cell line with a virus, wherein the avian cell line comprisesavian embryonic fibroblast cells or their progeny which areimmortalized, but untransformed and which comprise, in their genome, theSV40 T+t gene, allowing the virus to propagate, and harvesting thevirus.
 2. The method of claim 1, wherein the virus is an avian virus. 3.The method of claim 1, wherein the virus is a duck virus.
 4. The methodof claim 1, wherein the virus is chosen from the group consisting ofduck adenovirus, duck parvovirus and duck retrovirus.
 5. The method ofclaim 1, wherein the SV40 T+t gene is under the control of the MTIpromoter.
 6. The method of claim 1, wherein the cells comprise in theirgenome the SV40 promoter which is functionally linked to the gene forresistance to neomycin.
 7. The method of claim 1, wherein the cellscomprise in their genome at least one LTR sequence.
 8. The method ofclaim 1, wherein the cell line is cell line TDF-2A, which is depositedin the CNCM under reference number I-1712.